In yeast, the genes COX5a and COX5b encode structurally divergent, yet functionally interchangeable forms of subunit V of cytochrome c oxidase, a mitochondrial inner membrane protein complex. A form of this subunit, either Va or Vb, is required for cytochrome oxidase activity, and hence, for cellular respiration. In yeast cells, these genes are not expressed at equivalent levels; they are normally regulated in opposite ways at the level of transcription. This regulation leads to markedly different intracellular levels of subunits Va and Vb. We propose to continue, and to expand our work on the biology and regulation of the yeast COX5 genes. We will study, in detail, the molecular mechanisms that underlie the transcriptional regulation of these genes. In particular, we will focus on understanding how oxygen and heme exert an inverse effect on COX5 expression. This will be accomplished using a combined molecular, biochemical, and genetic approach. Using deletion, linker insertion, and site directed mutagenesis, we will continue to define the cis-acting elements involved in the transcription of the COX5a and COX5b genes. Using biochemical techniques, including gel-retardation assays and DNA footprinting, we will identify the trans-acting factors that interact with those specific regulatory sites. Using regulatory mutants, in conjunction with the biochemical assays already mentioned, we will identify and isolate the genes whose products are involved in COX5 expression. If successful, these studies will lead to a clear understanding of the regulation of the COX5 gene family. As such, they will contribute important insights into our overall knowledge of gene expression, of nuclear mitochondrial interactions, and of mitochondrial biogenesis.